1. Field of the Invention
Recent advances in the general field of molecular biology have made it possible to detect specific genes of clinical and commercial importance. For example, the structures of various genes and gene sequences associated with specific human diseases are known, as are various techniques for detecting the presence of such genes. It is therefore possible to diagnose human disease at the genetic level.
The most common technique for detecting a particular gene sequence is hybridization. A particular nucleotide sequence or "probe" is marked with a detectable label, typically a radioactive label or chemical modification, and combined with the nucleic acid sample of interest, either in situ as part of intact cells or as isolated DNA or RNA fragements. The sample can be either free in solution or immobilized on a solid substrate. If the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred and for measuring the amount of DNA/RNA sample present. The hybridization technique is of prime importance in basic research directed at understanding the relationship between nucleotide sequences and their function, as well as in diagnostic use to detect known aberrant genes or disease agents such as viruses or bacteria.
The main limitation of present gene detecting methods is that they are not sensitive enough and therefore require a relatively large amount of sample to accurately verify the existence of a particular gene sequence. This is not surprising since the detection of a single gene in the entire genetic repertoire of a human being requires locating one part in one to ten million. In fact, most hybridization methods require at least one in ten micrograms of purified DNA, representing a substantial sample of cells, to perform a reliable analysis. This limitation is particularly significant in pre-natal diagnosis of genetic disorders where only a small cell sample can be taken or to identify infectious agents such as viruses in small tissue samples. Consequently, there is a substantial interest in developing gene detecting methods which will increase the sensitivity of the hybridization assay without sacrificing its specificity.